Our long-term goal is to utilize biophysical methods to characterize the structure-function relationship of rhodopsin, using it as a template for understanding G-protein coupled receptors (GPCRs). GPCRs are major pharmaceutical targets because of the prominent role they play in cell signaling cascades. The specific aims of this project are to: 1) identify key interactions between rhodopsin and retinal in the meta II intermediate state;2) quantify the influence lipid bilayer forces exert on rhodopsin activation and oligomerization, and 3) use multiscale coarse grained molecular dynamics simulations to provide explanations for the role of GPCR oligomerization and lipid bilayer effects in rhodopsin activation. The methods chosen to achieve the specific aims provide a highly effective framework for complete understanding of rhodopsin structure and function. 2H NMR studies of rhodopsin in the meta II state have the advantage of providing detailed dynamical information about retinal movements in the rhodopsin binding pocket, and will give insights into how the counterion switch in the binding pocket helps drive rhodopsin into meta II. FRET, EPR, and DEER are three spectroscopic methods that will examine rhodopsin association in the cell membrane. They are non- disruptive to rhodopsin structure, and provide excellent time-dependent observations of monomeric interactions in a native environment. This allows several different lipid bilayer compositions to be tested, leading to further characterization of lipid bilayer effects on rhodopsin activation and oligomerization. SAXS will be used an additional method for measuring oligomerization effects during activation;all methods for specific aim #2 shift our focus from the atomic level (rhodopsin binding pocket) to the macromolecular level (rhodopsin monomers in the lipid bilayer). Finally, molecular simulations can be used to mimic native rhodopsin and predict general behavior. Besides being able to comparatively model 2H NMR lineshapes and SAXS radial distribution functions, molecular simulations, such as multi-scale coarse-grained molecular dynamics, can further extend the scope of our investigation to the mesoscopic scale. This will generate qualitative and quantitative predictions of rhodopsin interactions in heterogeneous, native-like, lipid bilayers. PUBLIC HEALTH RELEVANCE: Our research is relevant to public health because GPCRs constitute almost one-half of pharmaceutical targets today. Complete understanding of the rhodopsin structure-function relationship will help develop applications to treat disorders such as retinitis pigmentosa.